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by sender cells inoculated at a spot. Their model predicted that receiver cells sufficiently
far from the sender cells would not have the LuxR regulated circuit activated due to a low
concentration of 3OC6HSL. Here, the wild-type LacI repressor would repress GFP expression.
In contrast, at a sufficiently close distance to the sender cells, the high concentration of
3OC6HSL in the medium would result in the expression of both the LacI
M1
and CI repressors.
Here, LacI
M1
will directly repress GFP, while CI would repress expression of the wild-type
LacI repressor. However, at an intermediate distance from the sender cells, the concentration
of 3OC6HSL would be sufficient to trigger the repression of wild-type LacI repressor by the
CI repressor, but not the repression of GFP by the LacI
M1
repressor, as the CI repressor is a
more potent repressor of expression when compared to LacI
M1
. Overall, this would result in
suppression of the wild-type LacI repressor only, which in turn would allow expression of
GFP. On the whole, the circuit would result in high expression of GFP from the receiver cells at
an intermediate distance from the sender cells.
To control the upper 3OC6HSL threshold at which GFP expression was repressed, the
authors engineered three variants of the
circuit, which determines the
3OC6HSL concentration at which GFP expression was repressed, inside the receiver cells.
Each different
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circuit contains a LuxR protein with a different sensitivity
threshold to the 3OC6HSL signal. When combined with the
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circuit, which
determines the lowest concentration of 3OC6HSL that drives GFP expression, the authors
created three receiver cell strains: BD1 (high sensitivity to 3OC6HSL); BD2 (wild-type
sensitivity to 3OC6HSL, which was labeled with a red fluorescent protein (RFP), instead
of GFP); and BD3 (low sensitivity to 3OC6HSL).
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Using simulations, the authors predicted that by combining various BD receiver strains,
and the sender population, they could produce different patterns. Guided by modeling,
the authors experimentally created a bulls-eye pattern by plating a mixture of BD3 and
BD2 cells on an agar plate and by placing a disk containing sender cells in the middle.
The BD3 cells formed a GFP ring near the sender population, whereas the BD2 cells
produced a red ring farther away. Similarly, by coculturing BD1 and BD2 cells on an
agar plate, and in the presence of sender cells, a GFP ring formed outside of the RFP ring.
By varying the positioning of the sender cell population within a lawn of receiver cells,
the authors generated a wide variety of patterns including a four-leaf clover, an ellipse,
and a heart.
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ENGINEERED COOPERATION IN SYNTHETIC MICROBIAL
CONSORTIA
Auxotrophic strains have often been used to create cooperative synthetic consortia
(e.g.
21
which will be discussed later). Recently, Wintermute and Silver examined the
properties that led to efficient metabolite exchange, and thus cooperation, between pairs
of auxotrophic strains.
22
The authors tested 46 engineered, conditional lethal
E. coli
auxotrophs to determine if coculturing pairs of auxotrophs could lead to cooperation.
Each auxotrophic strain carries a mutation that abolishes its ability to synthesize an essential
metabolite. The metabolites range from amino acids to enzymes involved in cellular
respiration. When pairs of auxotrophs were cocultured, a subset of the cocultured strains
resulted in varying degrees of cooperation. For example, when an auxotrophic population
lacking
panD
(required for biosynthesis of pantothenate) and an auxotrophic population
lacking
proC
(required for proline biosynthesis) were grown in coculture, the
panD
auxotroph grew considerably, whereas the
proC
auxotroph did not. In contrast, when a
metA
auxotroph (required for methionine biosynthesis) population and a
lysA
auxotroph
(required for lysine biosynthesis) population were cocultured, both populations
grew markedly (
Fig. 13.1b
). A single
universal cooperator
could not be identified.
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